Publications

• Mutational biosynthesis of novel rapamycins by a strain of Streptomyces hygroscopicus NRRL 5491 disrupted in rapL, encoding a putative lysine cyclodeaminase.

  Khaw L.E., Bohm G.A., Metcalfe S., Staunton J., Leadlay P.F.

  The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by phiC31 phage-mediated gene replacement gave rise to a mutant which did not produce ... >> More

  The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by phiC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added L-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific L-lysine cyclodeaminase important for the production of the L-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle. << Less

  J. Bacteriol. 180:809-814(1998) [PubMed] [EuropePMC]

• Biochemical characterisation of recombinant Streptomyces pristinaespiralis L-lysine cyclodeaminase.

  Tsotsou G.E., Barbirato F.

  L-Lysine cyclodeaminase from Streptomyces pristinaespiralis was heterologously expressed in Escherichia coli, isolated to 90% purity after two purification steps and characterised. The size of the isolated recombinant enzyme was in agreement with the theoretical size calculated from the correspond ... >> More

  L-Lysine cyclodeaminase from Streptomyces pristinaespiralis was heterologously expressed in Escherichia coli, isolated to 90% purity after two purification steps and characterised. The size of the isolated recombinant enzyme was in agreement with the theoretical size calculated from the corresponding gene. We demonstrated that our preparation converts L-lysine to L-pipecolic acid (enantiomeric excess >95%) after isolating and identifying the conversion product by LC/MS, NMR and IR. This conversion followed Michaelis-Menten kinetics with a K(m) of 1.39+/-0.32 mM. The enzyme activity was maximal at pH 6.7. Reducing conditions, the presence of glycerol and in particular the presence of iron(II) significantly enhanced the L-lysine cyclodeaminase activity. Although the heat stability of the enzyme diminished significantly after 37 degrees C, the initial rate of reaction was maximal at 61 degrees C. We found no requirement for an external cofactor for full activity, although sequence data indicate NAD+ as cofactor. Upon enzyme denaturation, NAD+ release was observed, which indicates very tight binding of NAD+ to the enzyme. In parallel we developed selection and screening assays for lysine cyclodeaminase, which we adapted to microtitre plate format and validated. Among twenty-eight lysine analogues screened for turnover/binding to the enzyme, three were identified as substrates (L-ornithine, 5-hydroxylysine and L-4-thialysine), while another six (4-azalysine, L-2,4-diaminobutyric acid, 1,5-diaminopentane, N-epsilon-trifluoroacetyl-L-lysine, N-epsilon-Boc-L-lysine and N-epsilon-methyl-L-lysine) were shown to compete against L-lysine turnover without being converted by the enzyme. All substrates displayed Michaelis-Menten kinetics upon turnover by lysine cyclodeaminase. Our results indicate that the lysine cyclodeaminase from Streptomyces pristinaespiralis is a highly enantioselective enzyme at the substrate recognition and conversion levels, in both cases in favour of the l-isomer. << Less

  Biochimie 89:591-604(2007) [PubMed] [EuropePMC]

• Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

  Gatto G.J. Jr., Boyne M.T. II, Kelleher N.L., Walsh C.T.

  Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macroc ... >> More

  Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner. << Less

  J. Am. Chem. Soc. 128:3838-3847(2006) [PubMed] [EuropePMC]